CN104568876A - Method using thin-layered graphene fluorescence quenching combined with nano-metal array to perform single-molecule fluorescence observation - Google Patents
Method using thin-layered graphene fluorescence quenching combined with nano-metal array to perform single-molecule fluorescence observation Download PDFInfo
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Abstract
The invention belongs to the technical field of biophysical single-molecule observation surface treatment, and particularly discloses a method using thin-layered graphene fluorescence quenching combined with a nano-metal array to perform single-molecule fluorescence observation. The method comprises the following specific steps: processing the surface of a single-molecule fluorescence observation area; preparing a nano-metal ordered sequence on a graphene substrate. According to the method disclosed by the invention, the properties of the thin-layered graphene, such as quenching fluorescent molecules in a certain area of the surface, having good permeability, and enabling the loss of exciting light and fluorescence signals to be lowered, are utilized; the metal nano-structure on the thin-layered graphene can be normally combined with the fluorescent molecules, and the metal layer longitudinally lengthens the distance between the fluorescent molecules and graphene, so that the fluorescent molecules can normally generate fluorescence beyond the graphene quenching range; the concentration and the joining condition of the fluorescent molecules can be controlled to realize single-molecule fluorescence observation.
Description
Technical field
The invention belongs to biophysics unimolecule observation technical field of surface, be specifically related to a kind of single molecular fluorescence observation procedure.
Background technology
Single molecule experiments is the observation technology that current biologic applications aspect gets most of the attention, such as, utilize fluorescent dye or fluorescent protein labeling and observe the methods such as single target molecule, can obtain over and be averaged in polymolecular experiment and the information that falls into oblivion.But, the observation of transmission-type unimolecule to be realized so far encounter very large problem on the nano-structure array that glass is substrate.As a rule, the transmission-type single molecular fluorescence observation on nano-structure array be carried out, need to possess following condition: 1) substrate is transparent, can not too much loss exciting light and fluorescence signal, thus complete transmission-type and observe; 2) need effects on surface to carry out processing thus avoid fluorescence molecule non-specific binding in other regions except target location (nano-structure array), affecting single molecular fluorescence observation; 3) the process operation of effects on surface zones of different can not be disturbed (existing structure that such as can not destroy other regions for increasing specific binding efficiency to the surface treatment that target location is carried out) mutually.
For meeting Article 1 requirement, usually glass is selected to test as substrate, corresponding in order to prevent the combination of nonspecific fluorescence molecule, usually use the polymkeric substance such as the polyglycol after modifying, form the film that can prevent fluorescence molecule from combining at glass surface by the mode of self assembly.But such mode is also faced with some problems: 1. first the condition of polymer molecule film forming is comparatively harsh, the factors such as environmental pH, temperature, concentration all can affect the quality of its film forming, thus affect its function stoping combination; 2., secondly, when further processing and fabricating nano-structure array, the process for array surface is difficult to realize because thin polymer film is easily destroyed usually.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, propose a kind of high efficiency single molecular fluorescence observation procedure.
The single molecular fluorescence observation procedure that the present invention proposes, use thin graphene is substrate, combining nano metal array, can not affect modifying again nano metal while the fluorescence molecule of quencher non-specific binding, thus carries out high efficiency single molecular fluorescence observation.Concrete steps are:
(1) process on surface, single molecular fluorescence observation area:
A () selects glass flake (general glass thickness is about 0.15-0.20mm), carry out surface clean with the concentrated sulphuric acid and hydrogen peroxide potpourri (both weight ratios general are 2:1 to 4:1) to it;
B () dries up glass flake with high pure nitrogen, then use adhesive tape that Graphene is transferred to surface of glass slide, in surface of glass slide, some is thin layer (its thickness is less than 3 layers of atomic layer) to the Graphene of transfer;
(2) on above-mentioned graphene substrate, nano metal oldered array is prepared:
A () is in above-mentioned surface of glass slide, with spin-coating method coating glue-line PMMA(and polymethylmethacrylate), on glue-line, the position of thin graphene is marked under optical microscope, with electronic beam method AM aluminum metallization film (thickness is 5-10nm), then carry out electron beam exposure (EBL) in thin graphene position, write out the nano-structure array of design;
B () soaks the article through above-mentioned process with chemical solution, remove aluminium film, chemical solution used is the mixed liquor of 3038 developer for positive photoresist and deionized water, and the weight ratio of 3038 developer for positive photoresist and deionized water is 1:3 to 1:5;
C () removes PMMA with developer solution is aobvious: developer solution is chilled to 0-4 DEG C in advance, and low temperature develops about 30-40min, and developer solution used is the mixed liquor of MIBK and IPA, and in mixed liquor, the weight ratio of MIBK and IPA is 1:2 to 1:4;
D () with electronic beam method evaporation one metal level (concrete material depend on test the fluorescent material selected and using method, be generally gold or silver), in order to separate fluorescence molecule and Graphene, this metal layer thickness is 15-20nm, is preferably about 18nm; Soak with acetone again, the metal film that ultrasonic removing is unnecessary and PMMA;
Take pictures under (e) Electronic Speculum, determine the situations such as array integrality.
Thin graphene can fluorescence molecule in the certain area of quencher surface and itself is more transparent, can not the too much exciting light of loss and fluorescence signal.Fluorescence molecule can normally be combined on the nano metal array on graphene substrate, and because normally not sending fluorescence within the scope of Graphene quencher.Can realize single molecular fluorescence observation by control fluorescence molecule concentration and conjugation condition, concrete observation embodiment as shown in Figure 4.
Experiment shows, Graphene thickness be less than 3 layers and metal layer thickness is more than or equal to 18nm time, the fluorescence molecule that nanostructured is adsorbed by Graphene quencher, normally can not can carry out unimolecule observation.Observed pattern as shown in Figure 4.
Accompanying drawing explanation
Fig. 1 is the shape appearance figure of Graphene.Wherein, left figure is the shape appearance figure (in figure, the part of arrows has single-layer graphene to cover) of single-layer graphene under light field, and right figure is the image observed under TIRF.
Figure 2 shows that the observations under TIRF.Wherein, in a and b, arrow instruction is Graphene fragment, c is IFP1.4 same concentration lower-glass surface/(3nm Cr+15nm Au) surface/(Graphene fragment+3nm Cr+15nm Au) surface point other fluorescence intensity statistical value, the fluorescence intensity mean value at d schemes each group histogram (adjacent striped cylindricality and full packing cylindricality) the Graphene fragment place that is same position and neighbouring only layer gold place, Graphene thickness of each group there are differences.
Fig. 3 is nm of gold array of structures diagram prepared by thin graphene surface.Wherein, a is the array position light field schematic diagram after about 40 times of lower micro-nanos of amplification complete, the part that in figure, square circle goes out is nano-array position, b figure is circle delineation region enlarged image in the secure execution mode (sem in a figure, and it is the enlarged image of 1um and 200nm that c figure and d figure is respectively resolution under SEM.
Fig. 4 is unimolecule observed pattern diagram.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1: Graphene is to Quenching of fluorescence
1, the glass surface of glass slide after first extremely being cleaned by chemical gaseous phase depositing process transfer single-layer graphene, Graphene area coverage is about 1cm
2;
2, add fluorescin solution (used herein is IFP1.4 fluorescin, and concentration is about 400nM) at graphenic surface, observe under utilizing total internal reflection fluorescence microscope (TIRF) after rinsing out unnecessary fluorescin.
As shown in Figure 1, left figure is the shape appearance figure (part that in figure, red arrow indicates has single-layer graphene to cover) of single-layer graphene under light field, and right figure is the image observed under TIRF.As can be seen from right figure, the place having single-layer graphene to cover does not have obvious fluorescence signal, illustrates and directly sticks to fluorescence molecule on Graphene by quencher, cannot send fluorescence.
Embodiment 2: Graphene thickness is on the impact of fluorescent quenching
1, first by adhesive tape Graphene is transferred to cleaned after slide on;
2, evaporation 3nm Cr+15nm Au on the slide after step 1 processes, in order to separate fluorescence molecule and Graphene;
3, on slide, add the solution (concentration is about 400nM) of IFP1.4, a period of time backlash washes away unnecessary fluorescence molecule, and under TIRF, observation has the region of Graphene fragment.
Figure 2 shows that the observations under TIRF, in a and b figure, red arrow instruction is Graphene fragment, and the fluorescence signal of IFP1.4 still obviously can be observed in the region that this skim fragment covers as seen, illustrates that thin graphene does not affect Fluirescence observation.C figure is IFP1.4 same concentration lower-glass surface/(3nm Cr+15nm Au) surface/(Graphene fragment+3nm Cr+15nm Au) surface point other fluorescence intensity statistical value, can find out that the mean value of the fluorescence intensity on the Graphene fragment of different-thickness is really lower than the control group only having chromium layer gold slide, but (each group black and red histogram are the Graphene fragment place of same position and the fluorescence intensity mean value at neighbouring only layer gold place to d figure from behind, the Graphene thickness of each group there are differences) can find out, the Graphene fragment of different-thickness is different for the quencher scope of fluorescent material, Graphene thicker can the scope of quench fluorescence larger, the corresponding fluorescence intensity observed is less.And thin graphene can the scope of quencher less, thus the fluorescence intensity observed comparatively large (namely corresponding to the Graphene thin layer shown in a and b figure as d schemes one group of Graphene thin layer that blue frame goes out).
Embodiment 3: prepare nm of gold array of structures on Graphene thin layer, for single molecular fluorescence observation experiment
1, first select glass flake (thickness is about 0.17mm only), carry out surface clean with the concentrated sulphuric acid and hydrogen peroxide potpourri (7:3);
2, after high pure nitrogen dries up, use adhesive tape that Graphene is transferred to surface of glass slide, in surface of glass slide, some is thin layer (being less than 3 layer thicknesses) to the Graphene of transfer;
3, at surface of glass slide spin coating (PMMA), on glue-line, under optical microscope, mark the position of thin graphene, after electron beam evaporation plating aluminium film (about 7nm), carry out the nano-structure array that electron beam exposure (EBL) writes out design in thin graphene position;
4, in chemical solution, aluminium film is removed in immersion, and chemical solution is 3038 developer for positive photoresist: deionized water=1:4
5, aobviously in developer solution remove PMMA, developer solution need be chilled to 0 DEG C in advance, and low temperature develops about 30-40min, and developer solution is MIBK:IPA=1:3;
6, electron beam evaporation plating 3nm Cr+15nm Au, acetone soaks the unnecessary PMMA of ultrasonic removing;
7, to take pictures under Electronic Speculum situations such as determining array integrality.
Nm of gold array of structures prepared by thin graphene surface is shown in Fig. 3.B figure draws a circle to approve region enlarged image in the secure execution mode (sem in a figure, c figure and d figure is that under SEM, resolution is the enlarged image of 1um and 200nm respectively.Result shows the array of structures completely can preparing hundred nanometer scale on Graphene.
Claims (2)
1. a thin graphene fluorescent quenching combining nano metal array carries out the method for single molecular fluorescence observation, it is characterized in that: use thin graphene is substrate, combining nano metal array, can not modifying again nano metal be affected while the fluorescence molecule of quencher non-specific binding, thus carry out high efficiency single molecular fluorescence observation; Concrete steps are:
(1) process on surface, single molecular fluorescence observation area:
A () selects glass flake, carry out surface clean with the concentrated sulphuric acid and hydrogen peroxide potpourri to it;
B () dries up glass flake with high pure nitrogen, then use adhesive tape that Graphene is transferred to surface of glass slide, in surface of glass slide, some is thin layer to the Graphene of transfer, and its thickness is less than 3 atomic layers;
(2) on above-mentioned graphene substrate, nano metal oldered array is prepared:
A () is in above-mentioned surface of glass slide, with spin-coating method coating glue-line PMMA, under optical microscope, on glue-line, mark the position of thin graphene, with electronic beam method AM aluminum metallization film, then carry out electron beam exposure in thin graphene position, write out the nano-structure array of design;
B () soaks the article through above-mentioned process with chemical solution, remove aluminium film, chemical solution used is the mixed liquor of 3038 developer for positive photoresist and deionized water, and the weight ratio of 3038 developer for positive photoresist and deionized water is 1:3 to 1:5;
C () removes PMMA with developer solution is aobvious: developer solution is chilled to 0-4 DEG C in advance, low temperature development 30-40min, and developer solution used is the mixed liquor of MIBK and IPA, and in mixed liquor, the weight ratio of MIBK and IPA is 1:2 to 1:4;
D () uses electronic beam method evaporation one metal level, this metal layer thickness is 15-20nm; Soak with acetone again, the metal film that ultrasonic removing is unnecessary and PMMA;
Take pictures under (e) Electronic Speculum, determine array integrality situation.
2. method according to claim 1, is characterized in that described metal layer material is for gold or silver-colored.
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| CN105548131A (en) * | 2016-03-03 | 2016-05-04 | 中国烟草总公司郑州烟草研究院 | Preparation method of array fluorescent nano-cluster sensor and application of array fluorescent nano-cluster sensor to metal ion recognition |
| CN105806815A (en) * | 2016-03-14 | 2016-07-27 | 首都师范大学 | Fluorescent nanometer probe for detecting hydrogen sulfide and preparation method and application of fluorescent nanometer probe |
| CN108033438A (en) * | 2017-12-28 | 2018-05-15 | 中国华能集团公司 | One kind visualization carbon material structure and preparation method thereof |
| CN109540854A (en) * | 2018-11-03 | 2019-03-29 | 复旦大学 | Enhance the nanometer metal structure and preparation method thereof of near infrared band fluorescence signal |
| WO2021055688A1 (en) * | 2019-09-19 | 2021-03-25 | Northwestern University | Methods for imaging transparent two-dimensional sheets by fluorescence quenching microscopy |
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| CN105806815B (en) * | 2016-03-14 | 2019-04-05 | 首都师范大学 | A kind of fluorescent nano probe and the preparation method and application thereof detecting hydrogen sulfide |
| CN108033438A (en) * | 2017-12-28 | 2018-05-15 | 中国华能集团公司 | One kind visualization carbon material structure and preparation method thereof |
| CN109540854A (en) * | 2018-11-03 | 2019-03-29 | 复旦大学 | Enhance the nanometer metal structure and preparation method thereof of near infrared band fluorescence signal |
| WO2021055688A1 (en) * | 2019-09-19 | 2021-03-25 | Northwestern University | Methods for imaging transparent two-dimensional sheets by fluorescence quenching microscopy |
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